PROJECT SUMMARY: The SCORE grant will combine the PI expertise in protein encapsulation within silica based sol-gel matrices and nanoparticles with his work with stable cyclic nitroxide radicals and his expertise in spectroscopic methods and allow him to develop and apply it to address important challenges in public health. The objective is to generate novel nitroxides hybrid nanogels that will serve as non-toxic contrast agents for MRI diagnostic tests replacing the commonly used gadolinium-based contrast agents (GBCA) that are linked to the development of nephrogenic systemic fibrosis in patients. Our approach is innovative in that we utilize the sol-gel nanoparticle matrix to enhance the relaxivity of embedded nitroxides radicals as well as to increase their in vivo stability and inhibit their bioreduction. This research will explore methods for generation of sol-gel based hybrid nanoparticles with entrapped nitroxides or nitroxide-protein conjugates resulting in improved biostability, hydrophilicity and 1H water relaxivity (r1). In collaboration with Dr. Krishna, a leading expert in the field, select nitroxide hybrid nanogels (NHN) exhibiting improved activity will be characterized and optimized using in vitro MRI. These studies will identify promising NHN that will be further extended to animal studies at NCI. Specific Aim 1. Fabrication of Sol-gel based nanoparticles (nanogels): We will use tetramethylorthosilicate and/or tetraethylorthosilicate and their derivatives for sol-gel synthesis optimizing the nanogels size and solubility. We will explore an emerging biomimetic approach for the fabrication of hybrid nanogels. Specific Aim 2. Immobilization of nitroxides and nitroxide-protein conjugates within the nanogels. Selected nitroxides will be tested including TEMPOL, 3-CP, TEMPO-Lomustine, spirocyclohexyl nitroxide 1-OH and tetraethyl nitroxide 2-OH sterically protected nitroxides that are less susceptible to bio-reduction, as well as nitroxide-protein conjugates. Specific Aim 3. Structural and functional characterization of the entrapped nitroxides: We will study the size distribution, solubility, aggregation and loading capacity of the modified nanogel as well as rates of their release/leakage. UV/vis spectroscopy, EPR spectroscopy, and biochemical assays will be used to characterize the embedded nitroxides, including determination of their reduction rates. Specific Aim 4. Testing of the nitroxide entrapped hybrid nanogels as contrast agents for MRI in vitro and in mouse models. Optimize the NHN for highest relaxivity (>5mM-1S-1) in vitro followed by in vivo pharmacokinetic and toxicity characterization. This grant will serve to enhance the PI's research program, through acceleration of data collection and an increase in publication productivity, the PI will develop his research competitiveness and transition to non-SCORE support. The results of this research will be used to apply to an NIH RO1 grant by the third year of the project. Queens College undergraduate and graduate students, many of which are from populations under-represented in the biomedical research community, could participate in the research. Students will be exposed to methods and instrumentation of the emerging fields of bio-nanotechnology and nanomedicine, thereby enhancing their biomedical sciences skills. This research will overcome a public health challenge by developing non-toxic MRI contrast agents replacing GBCA that are linked to nephrogenic systemic fibrosis.